Autoclavable annular chromatograph

ABSTRACT

The invention relates to a CAC device comprising a stationary column head, a stationary eluate collecting ring, a rotating body which is located between said column head and said collecting ring and which can rotate about a vertical axis and contains a chromatographic separating medium; and a drive device. Said rotating body ( 1 ) is connected to a rotating platform ( 4 ) at its bottom end. This rotating platform ( 4 ) is slidingly, sealingly guided on the eluate collecting ring ( 5 ) and has an extension on the underside. The extension can be coupled to a drive shaft ( 2 ) of the drive device ( 8 ) with a rapidly detachable connection and the stationary column head the rotating body ( 1 ) with the rotating platform ( 4 ) and the attached eluate collecting ring ( 5 ) can be connected to form a CAC module and can be removed from the drive shaft ( 2 ) as a unit. The invention also relates to an inverse CAC method using the inventive device. According to this method, the sample material is distributed over the entire periphery of the separating medium.

[0001] The invention relates to a device for continuous annularchromatography sterilizable in an autoclave, and to a method for usingthe device.

[0002] From WO 99/12625, an annular chromatograph is known which has astationary eluate collecting ring formed as a slide ring on which arotating body comprising the chromatographic gel particle bed in theshape of a cylinder jacket and an integrated bottom plate is slidinglyand sealingly guided. The purpose being to protect continuous annularchromatography (international abbreviation CAC) performed with thisdevice against contamination by the environment and to enable sterilework. Collecting points in the form of chambers with liquid withdrawalare arranged in the eluate collecting ring. Channels receiving thefractions draining from the particle bed of the CAC column and conveyingthem to the chambers of the eluate collecting ring are present in thebottom plate.

[0003] Particularly for separations of biological or biochemicalmaterials and in applications for the working up of pharmaceuticallyactive substances, the requirements for a chromatographic process freeof contamination are stringent. Systems closed toward the outside arerequired here which are readily sterilized and in addition are readilyadapted to changing separation jobs, while it must be remembered thatthe device that is to be operated free of contamination consists ofrotating and stationary parts. In addition, a need exists for provisionsbeing taken during eluate withdrawal that the discharge of the eluate orfractions from the particle bed and the chambers in the collecting ringbe as uniform as possible and at the same time the separation as sharpas possible. Moreover, appropriate measures should be taken so thatfractions already separated will not later be “spread” to neighboringchambers.

[0004] According to WO 99/12625, the desire for improved discriminationin the separation was met by design, by raising the number of channelsin the bottom plate and of chambers in the collecting ring to a maximumthat was technically feasible and represented an economically meaningfulmaximum. The resulting problems caused by effects of adhesion andcapillarity due to the reduced cross sections of the channels in thebottom plate and particularly of the liquid withdrawal ducts of thecollecting ring chambers were solved by increasing the diameter of thechannels in the bottom plate radially upward toward the particle bed soas to make them wedge-shaped. The chambers were additionally providedwith pressure equalization ports that for sterile operation can becovered up by suitable sterile filter membranes, in order to prevent thedevelopment of underpressure and resulting aspiration of nonsterileforeign air into the chambers via the slide ring packing.

[0005] The undesired spreading of parts of the fractions intoneighboring chambers of the collecting ring was reduced according to WO99/12625, primarily by selecting a diameter of the channels in thebottom plate that was considerably larger than the thickness of theradial walls between the chambers of the collecting ring. The liquidstream arriving from a channel is then cut off by the radial walls aswith a shear blade when the channel slides over such a radial wall, sothat the liquid column existing between channel and chamber which isimportant for a regular liquid drain and which is substantially thickerthan the radial wall between two neighboring chambers, is interruptedonly in part but never over its entire cross section.

[0006] The present invention which primarily represents a development ofthe CAC device from WO 99/12625 but is not restricted to the features ofthis document refers to a device for annular chromatography with astationary column head, a stationary eluate collecting ring, a rotatingbody between head and ring which is rotatable about a vertical axis andconsists of a twin cylinder forming a hollow jacket filled with thechromatographic separation medium, and a mechanical drive. At its bottomend the rotating body is connected with a rotating platform which hasvertical through channels and is guided sealingly and slidingly on astationary eluate collecting ring with chambers. The rotating platformhas an extension at its underside that can be coupled with a drive shaftof the drive by a rapidly detachable connection. The stationary columnhead, the rotating body with the rotating platform and the adjacenteluate collecting ring are combined into a CAC module and can be liftedas a unit from the drive shaft.

[0007] The rotatable and stationary parts of the device according to theinvention form a unit that is independent of the drive and is heldtogether by suitable means. The rotating body and the stationary partsof the device without the drive unit can be clamped into a kind of cage,for instance between plates, in order to secure a durable union,particularly of the sliding seal between the rotating body and theeluate collecting ring, even during manipulations such as removal fromthe drive or transport.

[0008] It is the purpose of this measure to have a chromatography devicewhich together with the rotating body, the stationary column head andports for liquid feed lines, the eluate collecting ring, the liquiddrains of the chambers in the eluate collecting ring and, whereapplicable, the eluate collection vessel can be removed as a unit fromthe drive block, for instance, in order to sterilize it in an autoclave.However, the CAC device according to the invention preferably containsas well all design elements needed for a superheated-steam sterilizationin situ, in the manner known particularly from fermentation technology,by passing saturated steam while the device is attached to the driveblock. For a sterile process, preferably both the ducts feeding liquidto the column head and the ducts draining the chambers of the eluatecollecting ring are provided with sterile connectors. For the feedducts, for instance, standard sterile connectors such as the ones knownfrom fermentation technology can be used. Additional sealing can also beused, particularly at the seams or contact points between components ofthe rotating body.

[0009] Shafts, V-belts, driving chains, toothed-wheel gearing andfriction gear are suitable as a drive. The V-belt pulleys or gear rimscan be mounted directly at the periphery of the rotating body; toothedwheels and friction wheels can also act on the inner wall of therotating body having the shape of a cylinder jacket.

[0010] Drive shafts are preferred onto which the rotating body can beplaced and to which it can be coupled. It is preferred here to couplethe rotating body to the drive shaft, directly via a bottom plate shapedas a rotating platform immediately adjacent to the particle bed. To thisend, the rotating platform has a short extension at its underside whichis essentially of the same shape as the drive shaft and terminates intoa sleeve which can be aligned with and pushed over the drive shaft so asto accept the torque of the drive shaft. Known torsion connectors andsecurities against twisting of the shafts can be used for coupling.Rigid coupling connectors for aligned shafts or radial serrations canfor instance be used. What matters is that the rotating body can beremoved from the drive shaft in a reversible and simple fashion withoutany demanding assembly work, and that the torque is reliablytransmitted. Other coupling connectors suitable for connecting therotating body to the drive shaft are known to one skilled in the art,and include plug-in, snap, slide-lock, catch, and screw connections.

[0011] At its coupling end, the drive shaft can be shaped in knownfashion, for instance as a cylinder, cone, or polyhedron; the extensionof the rotating platform to be attached to it must then be provided withan aligned sleeve having a complementary shape. Conversely, the driveshaft can terminate in a sleeve into which the extension of the rotatingplatform is precisely fitted with its end having a complementary shape.

[0012] The slide ring seal between the rotating platform and thestationary eluate collecting ring must always be compressed as needed sothat the rotating body will be guided sealingly on the eluate collectingring. The rotating body's own weight can provide the needed pressure.However, it is preferred according to the invention that one or severalprestressed compression or tension springs are used. When prestressedtension springs are used, the eluate collecting ring is hung into them;when compression springs are used, they exert pressure from below on theeluate collecting ring. It is advantageous to provide pressure transferpads with a pressure surface larger than the springs between the springsand the eluate collecting ring in order to distribute the spring forceuniformly over the section of the eluate collecting ring.

[0013] The slide ring seal, the eluate collecting ring with its embeddedchambers receiving the eluates draining from the particle bed, as wellas the channels in the bottom plate or rotating platform can be realizedin the fashion described initially, and known from WO 99/12625. However,in the present invention it is preferred to solve the problem ofpressure fluctuations or underpressure described initally for the CACdevice of WO 99/12625 in another way than by using pressure equalizationports in the chambers of the collecting ring.

[0014] In fact, it has surprisingly be found that hoses used as eluatewithdrawal ducts from the chambers of the eluate collecting ring whichwhere appropriate can be pushed over outlet sockets present at thechambers will solve the underpressure problem even without pressureequalization ports in the chambers, provided the hoses coming from thechambers or outlet sockets are made into siphons, by being for instancebent into a wave shape similar to a recumbent “S”. It is unimportantthen whether the hoses or eventual outlet sockets are attached to thebottom of the chambers and essentially point vertically down, or whetherthey are laterally attached to the chambers and preferably point outwardand obliquely downward.

[0015] In this connection, it is decisive for optimum function of theCAC device according to the invention that between the particle bed andthe siphons of the draining hoses a liquid through connection as incommunicating vessels arises which despite rotation of the particle bedis preserved throughout the phases of elution and, where applicable, ofwashing and regeneration. For this reason it is not necessary either tokeep the ends or highest portions of the draining hoses at a level belowthe chambers of the collecting ring, they rather can—and preferablyshould—be lifted to a level higher than the collecting ring andmaintained there.

[0016] Apart from the invention being adapted for a sterilization in anautoclave, the modular structure of the device offers the advantage ofsimple interchangeability of the entire chromatography unit. Dependingon the type of separation problem being dealt with, rotating bodiesfully prepared can without waiting be placed one after the other ontothe drive as complete chromatography units (modules) so that during apreparative CAC process, another module can already, for instance, befilled and conditioned in parallel.

[0017] It is not only possible to operate one and the same drive blockwith alternating chromatography modules, one also can adapt the hollowcylinder jackets containing the separation medium and the eluate outletsto the particular chromatographic job. For instance, hollow cylinderjackets or rotating bodies can be varied in their length by using theappropriate spacer rods of different length. The invention is alsosuited for compound bodies where several particle beds on top of eachother or next to each other are used simultaneously for a separation,such as for instance described in WO 99/47913. By analogy, the basicidea and the principle of the present invention can be applied quitegenerally to all variants of particle beds and CAC devices such asdisclosed in the international patent applications WO 98/45699, WO99/12625, WO 99/29388, WO 99/28740, and WO 99/47913. The term “particlebed” should be understood here, not exclusively as a particulatechromatography gel or resin arranged in the hollow cylinder jacket ofthe rotating body but also as a monolithic separation medium suitablefor liquid chromatography, for instance made of ceramic, plastic (forinstance block polymer), fiber material and the like. The term alsorefers to a combination of two or several chromatographic separationmedia, where necessary in superimposed layers, preferably withseparating layers in between, such as known from the precitedpublications.

[0018] Below, the invention will be explained more closely in theinstance of an embodiment while referring to the drawings. It is shownin:

[0019]FIG. 1 a longitudinal section through the axis of rotation of aCAC device according to the invention;

[0020]FIG. 2 a detailed view of the bottom region of the device of FIG.1 with added representation of an eluate drain arranged in wave shape;and

[0021]FIG. 3 a graphical representation of the result of a concentratingprocess using the CAC device according to the invention.

[0022] In FIG. 1 a drive block 8 with drive shaft 2 connected with therotor 10 of the motor 9 can be recognized. A rotating body 1 is placedon the drive shaft 2.

[0023] The rotating body 1 is closed toward the outside and serves toperform annular chromatography. At the rotating body 1, a hollowcylinder jacket constituting the particle bed space 3 supported by arotating platform 4 is provided for receiving the particle bed. In theembodiment shown, the hollow cylinder jacket is placed upon the rotatingplatform 4 from above. As shown in this example, the inner cylinder ofthe hollow cylinder jacket can be connected below with a bottom support11, and through this support it can be fixed at the rotating platform 4via a screw connection 12, while above it can be closed off with aconical roof 13. For centering and stabilizing of the inner cylinder,the rotating platform 4 can have a central projection at its upper sidewhich is aligned and engages with a recess in the underside of thebottom support 11 which is of complementary shape. It is advantageousfor a contamination-free process to provide for additional seals 34 a,34 b, 34 c, 34 d in the seams between the contacting portions of thecomponents delimiting the particle bed space 3.

[0024] For torque transfer from the drive shaft 2, the rotating body 1can be coupled with the drive shaft 2. To this end the rotating platform4 has an extension at its underside which essentially has the shape of acentral portion of the drive shaft 2, and at its end terminates into asleeve 32 which can be aligned with and pushed over the free end ofdrive shaft 2 which by the lock-and-key principle has a shapecomplementary to that of sleeve 32. This coupling connection can besecured against twisting by a pin 33 laterally arranged in the driveshaft 2 and engaging into a corresponding recess of sleeve 32. Ofcourse, the opposite version where the drive shaft ends into a sleeveinto which an extension of the rotating platform 4 of complementaryshape is engaged will be suitable as well and serve the same purpose.

[0025] A retaining strip or grid 14 inserted into the rotating platform4 beneath the particle bed space 3 supports the particle bed andseparates it from the adjoining channels 15 which preferably areexpanded like wedges toward their upper ends, pass through the entirerotating platform 4, and convey the liquids draining from the particlebed to the chambers 16 (FIG. 2) of the eluate collecting ring 5. Fromthe bottoms of chambers 16 of the eluate collecting ring 5 which, wherenecessary, are tapering like funnels or are beveled toward the outside,draining channels 17 (FIG. 2) lead out of the eluate collecting ring,and eluate drains 18 (FIG. 2) which in general are plastic hoses areattached to them in order to provide the connections—sterile, wherenecessary—between the eluate collecting ring 5 and the eluate collectionvessels 19.

[0026] It is preferred to have eluate drains 18 laid out in wave shapelike a recumbent “S”, as shown in FIG. 2, in which case between a firstpart 18 a of the eluate drain 18 running downward and the adjoining part18 b bent upward, a siphon is formed while the part 18 c which followsnext and is once more bent downward ends in an eluate collection vessel19. This arrangement of the hoses of eluate drains 18 is readily broughtabout, for instance by lifting the hoses with a ring-shaped supportsurrounding the rotating body 1 which where necessary can be adjusted inits height (not shown in the figures).

[0027] When operating the CAC device as intended, between the particlebed space 3 and each eluate drain 18 a continuous liquid column isformed which is never completely broken because of the geometries ofchannels 15 in the rotating platform and of the radial walls betweenchambers 16 in the eluate collecting ring 5 which are specificallymatched as discussed at the outset in the instance of WO 99/12625. Bysiphon formation in the eluate drains 18 according to the invention, itis secured that the liquid column starting in the particle bed will atleast attain the siphon, and perhaps the upper bend of the wavelikeeluate drains 18 that follows after the siphon, and thus a regulareluate flow is maintained. At the same time the chambers 16 areprevented from emptying completely and an underpressure cannot arise bya possible abrupt emptying of the chambers. Minor pressure fluctuationswhich still might occur are buffered by the liquid column in the eluatedrains 18, so that an aspiration of (nonsterile) outside air through theslide ring seal cannot occur in any phase of the chromatographicoperation.

[0028] For eluate drainage without contamination, the rotating platform4 is guided slidingly and sealingly on two concentric gliding surfacesalong the periphery of the eluate collecting ring 5, the chambers 16being arranged between the gliding surfaces and the eluate collectingring 5 being pressed against the rotating platfrom 4 by a helical spring6. The helical spring 6 is centrally mounted so that the windings of thespring run around the axis of drive shaft 2. It transmits the springforce via a pressure pad 7 connected with the eluate collecting ring 5by a screw connection 12′ and acting as pressure transfer pad.

[0029] Between the inner gliding surface and the inner rim bordering thedrive shaft, the eluate collecting ring 5 has a peripheral recessbeveled toward the outside rim which with the underside of rotatingplatfrom 4 forms a hollow space 20; from the lowermost region of thisspace, one or several bores 21 lead from the eluate collecting ring 5 tothe outside. On one hand the hollow space serves to delimit the size ofthe gliding surface, on the other hand it serves to enablesuperheated-steam sterilization of the gliding surfaces. The bores 21then allow condensate accumulated during sterilization or duringchromatograph operation (for instance when cooling to temperatures belowroom temperature) to be drained from this space.

[0030] It should be pointed out here that the CAC device according tothe invention is preferably provided with means for temperature controland/or thermostatic operation, particularly with a heating and/orcooling circuit arranged inside and/or outside of the rotating body 1 inorder to be able to create optimum operating conditions by heating orcooling for each chromatographic task and starting material employed.Temperature control can for instance be achieved with a liquidtemperature control medium inside the inner cylinder of the rotatingbody 1 and/or by thermal insulation of the rotating body on its outside.The inner cylinder can also be realized as a twin jacket allowingcirculation of the temperature control medium.

[0031] The rotating body 1 with rotating platform 4, stationary columnhead, eluate collecting ring 5, and pressure pad 7 with helical spring 6is clamped between a torsionally rigid base plate 22 and a torsionallyrigid head plate 23. The plates are screw-connected via separator rods24 along their peripheries and are held at the desired distance by theserods. Approximately at the level of the rotating platform 4 along theseparator rods 24, a step is provided which supports an axial bearingshell 25 with O-ring seal 26 on which an annular bearing runner 25 aconnected via a screw connection 12″ with the rotating platform 4 issupported and guided slidingly.

[0032] The column head with supply ducts 27 a, 27 b, 27 for materialfeed, eluants, charging of particle bed material etc. having sterileconnectors known from fermentation technology is integrated into thehead plate 23 or connected with it as a single piece (as shown in theexample of FIG. 1). The top part of the rotating body 1 which closes offthe particle bed space 3 above is guided in the head plate 23 by afurther axial sliding bearing via an O-ring seal 26′ and annular bearingrunner 25 a′ connected by bolts; the axial bearing shell 25′ is solidlyattached to the torsionally rigid head plate 23.

[0033] The base plate 22 has a central opening with an internal diameterapproximately corresponding to the diameter of the drive shaft. Theedges of this central opening develop into a cylinder jacket 22 c risingvertically upward; its inner surface serves as guide sleeve for theextension 32 of the rotating platform 4. At the underside of base plate22, the opening is expanded to a cone 22 a substantially facilitatingplacing the CAC module onto the drive shaft 2. The cylinder jacket 22 cin turn is surrounded by a slide seal collar 28 facilitating a verticaladjustment of base plate 22 relative to pressure pad 7. A pin 29preventing the pressure pad 7 from turning relative to base plate 22 isslipped into the head portion of cylinder jacket 22 c. The pressure pad7 has a central through bore which in its diameter in a top portionadjacent to the eluate collecting ring 5 corresponds to the diameter ofextension 32, and in the diameter in its adjacent portion corresponds tothe diameter of cylinder jacket 22 c including the collar 28, so that itcan be pushed on one hand over the extension 32 of the rotating platform4 and on the other hand over the collar 28 and the cylinder jacket 22 cof the base plate. In a peripheral region 7 a of pressure pad 7, anannular recess opening downward is provided which accepts the helicalspring 6, the base plate 23 acting in this case as abutment for thehelical spring 6. The pressure pad 7 is held at the base plate 22 butnot fastened at it, hence the base plate 22 can be shifted or adjustedrelative to this pad along the collar 28 in a vertical direction so thatthe spring pressure and thus the compression of the slide ring sealbetween eluate collecting ring 5 and rotating platform 4 can in anyevent be varied. A peripheral projection 22 b projecting into theannular recess of cylinder jacket 7 a is provided at the bottom plate 22for further security. Finally, pressure pad 7 is solidly connected withthe eluate collecting ring by a screw connection 12′.

[0034] The CAC module according to the invention, with the CAC devicewith rotating body 1, eluate collecting ring 5, pressure pad 7 andhelical screw 6 clamped between base plate 22 and head plate 23, isplaced onto a support plate 30 of the drive block 8. A central borehaving its upper rim shaped as peripheral projection 30 a that iscomplementary to the adjacent cone 22 a of base plate 22 is provided inthe support plate 30 to allow the drive shaft 2 to pass through. Whenthe screw connection 31 between base plate 22 and support plate 30 isseparated, the entire CAC module can simply be lifted from the driveblock 8, where necessary with a crane, and autoclaved as such orreplaced by another CAC module.

[0035] The CAC device according to the invention is basically suited forall current separation problems amenable to CAC or P-CAC, particularlythose described for instance in WO 99/28740. This also includes the useof the most diverse chromatography gels or resins and combinations ofsuch resins and gels, such as also described for instance in WO99/28740. The use of monolithic separation media (for instance blockpolymer) or other separation media suited to liquid chromatography shallalso be included here. The CAC device according to the invention ispreferably employed for the purposes of preparative separation,purification and/or concentration of individual substances or mixturesof organic substances, particularly of proteins or protein mixtures ofvegetal, animal (including human), or biotechnological origin (forinstance, recombinant proteins) such as already quoted in part in WO99/28740.

[0036] The sample material to be treated is introduced as a feed intothe separation medium (for instance a particle bed of chromatographygel) at one point in the annular gap of the hollow cylinder jacket andconveyed by eluents (for instance chief eluent and step eluents) throughthe stationary phase, where adsorption and desorption processes canoccur. Commonly, the chief eluent is applied in uniform distributionover the entire annular gap, for instance via the conical roof coveringup the inner cylinder of the rotating body above, while the stepeluent(s) are also introduced at specific points located at particularangular distances from the sample feed point, and distributed over theannular gap. Apart from the supplies of feed and eluents, supplies ofconditioning liquids, particularly washing, regenerating, and/orsanitizing agents can be present which can be arranged in front of,between, and/or behind the eluent inlets, depending on the type ofprocessing job and sample material. This can be a considerable advantageor even a necessity when processing biological sample material oftenaccompanied by numerous undesirable foreign substances, on one hand, andhaving organic components (for instance proteins) which are perishableand susceptible to infections, on the other hand. This is all the moretrue when dealing with materials subsequently to be used for medicalpurposes in man or animals.

[0037] It has now been realized that in some separation, purification,and particularly concentration jobs a procedure is advantageous wherethe supplies of sample feed and chief eluent are interchanged ascompared with the traditional procedure, that is, by inverting thecommon approach, not the chief eluent but the sample material to betreated being applied while it is distributed over the entire annulargap. This approach makes sense, particularly when large volumes ofsample material must be processed, for instance when a pharmaceuticalactive substance present at a very low concentration in a solution mustbe concentrated and/or purified from foreign substances.

[0038] Such problems can be handled much more economically with this“inverse” CAC method (also called “head-space” method) than with thetraditional process variant, since the elution for instance can occur byeasy and rapid desorption in a small angular segment or a small zone.For a selective desorption of the sample material adsorbed at theseparation medium, one or several step eluent inlets and, whennecessary, further inlets for washing, regenerating, and/or sanitizingagents are arranged at predetermined angular distances in front of,between, and/or behind the eluent inlets. Here it must be observed incertain cases that with elution and/or conditioning steps (for instancewashing, regenerating, and/or sanitizing steps) to be performed indirect succession the corresponding supplies of elution and/orconditioning agents are arranged so close together that in between,sample material cannot enter the separation medium. As a complement oralternative, the flow rates of the sample-feed and detergent streams canbe so adjusted relative to each other that the same effect is achieved.

[0039] It is preferred to this end to provide the column head with amultitude of extension tubes for the liquid feeds which are permanentlymounted at short angular distances from each other. Of these, only apart is used and the others are closed off, depending on the separationtask.

[0040] As an alternative, in another embodiment a smaller number ofextension tubes are attached in mobile fashion at a guide ring (notshown in the figures) or annular guide rail above the annular gap of thehollow cylinder jacket, so that they can be shifted along the peripheryof the ring or guide rail and fastened at the desired angular positionsover the particle bed. To this end the extension tubes can be straighttubes. They preferably pass vertically through the guide ring or guiderail and are supported by glide or roll elements which are known forinstance from sliding bearing technology and are fixed to the tubes.

[0041] For a vertical stabilization and immovable fixation at the guidering or guide rail, the tubes can be tightened by screw or clampconnections beneath or above the guide ring or guide rail, or on bothsides. In an analogous manner, other suitable suspension systems can beused for the tubes, so long as they allow the tubes to be shifted alongthe periphery of the annular gap above the particle bed. The tubes forinstance can also be suspended in a mobile fashion with the aid of ringsor ears at a single guide ring or at two superimposed guide rings, andfastened with suitable clamp connections. The guide rings or rails canalso have locking positions where the tubes can be fixed in a stationaryway. The connection between the tubes and the inlet ports in the columnhead is provided by flexible hoses of an inert material (for instanceplastic), which may when necessary be armored, and can be attached tothe tubes and the inlet ports in the column head by sleeves, plug-in,snap, or screw seals or similar detachable connecting means.

[0042] The optimum conditions for selecting or adjusting the angularpositions, angular distances of the extension tubes, and/or flow ratesof the liquid streams supplied can be determined by preliminaryexperiments for all preparative processing problems. This will beillustrated with the example following below.

EXAMPLE Concentration of a Protein Solution by Inverse P-CAC

[0043] A solution essentially free of particles obtained from afermentation for the microbial production of recombinant human factorVIII (abbreviated: rFVIII), for concentration of the rFVIII contained init, is subjected to preparative continuous annular chromatography(P-CAC) using the CAC device according to the invention.

[0044] A CAC device is used which has a column head with seven inlets: amain inlet which distributes the sample material via the conical roof ofthe inner cylinder over the entire annular gap of the particle bed(except for those zones where elution or conditioning agents areintroduced), and when necessary fills the entire head space of therotating body with sample material, and one inlet each for washing andequilibrating solution (150 mM NaCl), eluent (0.7 M NaCl), regeneratingagent (2 M NaCl), detergent (sodium lauryl sulfate), and sanitizingagent (1 M NaOH); in this example, one inlet remains idle.

[0045] A Poros DEAE anion exchanger gel (maker: Perseptive Biosystems)is used as the particle bed in the hollow cylinder jacket of therotating body, and covered with a layer of glass beads about 2-3 cmthick. All inlets except the main inlet are provided with appropriatelybent extension tubes immersed into the glass bead layer and terminatingabout 0.5 to 1.5 cm above the gel bed of the anion exchanger. It isprevented in this way that the liquid streams discharged from theextension tubes directly “shoot” onto the gel bed and slurry up itsuppermost region. Yet even when using glass beads as a protective layerand application aid, the flow rates of the liquids flowing through theextension tubes directly into the annular gap are relatively narrowlylimited, so the introduction of the sample feed via one of these tubeswould represent a rate-determining step. The problem was solved by theflooding of the head space with sample feed as provided by theinvention.

[0046] For sterilization, the CAC module which has been fully chargedand filled with deionized water or, when applicable, a washing liquid,is pinched off at the liquid feed ducts of the column head, orpreferably tightly closed off with autoclavable membranes, particularlyin the form of corresponding screw or plug-in connectors as known fromfermentation technology. The hoses of the eluate drains of thecollecting ring, preferably numbering between 90 and 180, are alsotightly pinched off at their ends. They can also be provided with metalsyringes facilitating an aseptic connection of the hoses with sterilizedeluate collection vessels, where applicable containing a sealingmembrane, for instance by piercing these membranes under flameprotection. For the purposes of autoclaving, the syringes can inaddition be protected by reusable protecting caps so as to furtherreduce the danger of contamination.

[0047] The stationary head plate closing off the rotating body abovecontains at least one more opening serving to enable a pressureequalization in the rotating body during autoclaving and thereafter inthe phase of cooling. To this end the opening is covered by a sterilefilter membrane which is autoclavable and permeable to air and humidityin both directions, for instance with a pore size of at most 0.45 μm, oralternatively with an in-depth filter system (for instance glass wool).Such pressure equalization systems are also sufficiently well known fromfermentation technology. For autoclaving, the entire CAC module afterunscrewing the screw(s) between base plate and rotating platform islifted from the drive shaft of the motor drive, when necessary with amobile crane, and placed into an autoclave of suitable size. Theautoclaving which follows is performed in known fashion. For mostpractical applications, an exposure of 15 to 20 min at 120° C. (at apressure of about 0.15 MPa or about 1.5 atm) will suffice.

The P-CAC Process

[0048] After autoclaving, cooling, and aseptic connection of the feedlines to the pumps, and of the eluate drain lines to the eluatecollection vessels, preparative continuous annular chromatography wasstarted by supplying the sample feed with a flow rate of 80 ml/min. Thefive inlet extension tubes for washing/equilibration, elution,regeneration, cleaning, and sanitizing were arranged so that theyterminated in the glass bead layer of the annular gap within a singleangular segment of 45°, thus hindering or completely preventing apenetration of sample material into the separation medium within therange of these angular positions. In this way it was guaranteed thatbehind the elution zone and between the conditioning zones, no samplematerial will contaminate the separation medium.

[0049] The flow rate for washing/equilibration was set to 4 ml/min, theother flow rates were each set to 2 ml/min. An angular velocity of 120deg/h was selected as the rate of rotation for the rotating body.Sampling occurred after about 1.25 h. Despite processing conditionswhich were not yet optimum, a concentration of rFVIII by a factor of 13was achieved (FIG. 3).

[0050] In another experiment the flow rates were changed, so that thesample feed occurred with 100 ml/min, washing/equilibration and elutionwith 3 ml/min each, regeneration, cleaning, and sanitizing with 2 ml/mineach. At an angular velocity of 120 deg/h and a linear flow rate of 157cm/h, collection of the fractions was started after 3 h. A concentrationof the protein by a factor of 74 relative to the starting solution couldbe achieved (Table 1). TABLE 1 Concentration of rFVIII by P-CAC Drainposition 1 2 3 4 5 6 7 8 9 Titer 0 74 24 9 0.3 2.3 0.3 0 0 [E/ml]

1. Device for continuous annular chromatography (CAC) with a stationarycolumn head, a stationary eluate collecting ring, between them arotating body rotatable about a vertical axis and shaped as a twincylinder forming a hollow jacket with a chromatographic separationmedium contained therein, as well as a drive, the rotating body (1)being connected at its bottom end with a rotating platform (4) havingvertical through channels (15) which is guided sealingly and slidinglyon a stationary eluate collecting ring (5) with chambers (16),characterized in that the rotating platform (4) at its underside isprovided with an extension that can be coupled to a drive shaft (2) ofthe drive (8) via a rapidly detachable connection, and the stationarycolumn head, the rotating body (1) with rotating platform (4) and theadjacent eluate collecting ring (5) are combined into a CAC module andcan be detached as a unit from the drive shaft (2).
 2. Device accordingto claim 1, characterized in that at its end the extension of therotating platform (4) is shaped into a sleeve (32) into which an end ofthe drive shaft (2) that is of complementary shape is engaged, orconversely, in that the drive shaft (2) at its end is provided with asleeve into which an extension of the rotating platform (4) that is ofcomplementary shape is engaged.
 3. Device according to claim 1 or 2,characterized in that the column head, the rotating body (1) withrotating platform (4), and the eluate collecting ring (5) are clampedbetween a base plate (22) and a head plate (23), the base plate (22) andhead plate (23) where applicable being held together by distance rods(24) arranged concentrically around the rotating body (1).
 4. Deviceaccording to claim 3, characterized in that the column head has at leastone liquid supply line (27) and preferably is integrated into the headplate (23) or is connected with it as a single piece.
 5. Deviceaccording to one of the preceding claims, characterized in that therotating platform (4) is supported by a peripheral axial bearing (25)borne by distance rods (24), and is slidingly guided on this bearing,preferably via a horizontal O-ring.
 6. Device according to one of thepreceding claims, characterized in that a pressure pad (7) pushing theeluate collecting ring (5) against the rotating platform (4) is arrangedadjacent to the underside of the eluate collecting ring (5).
 7. Deviceaccording to claim 6, characterized in that the pressure pad (7) in aperipheral region in a peripheral recess (7 a) opening downward has ahelical spring (6) acting as a compression spring wound concentricallyaround the drive shaft (2) and abutting the bottom plate (22) arrangeddirectly beneath.
 8. Device according to one of the preceding claims,characterized in that the rotating platform (4) rests on two concentricgliding surfaces on the eluate collecting ring (5) and the eluatecollecting ring (5) between the inner gliding surface and its inner rimadjoining the drive shaft (2) has a recess beveled toward the outsidewhich together with the rotating platform (4) forms a hollow space (20)from the lowest point of which at least one bore (21) leads from theeluate collecting ring (5) to the outside.
 9. Device according to one ofthe preceding claims, characterized in that the eluate collecting ring(5) has a multitude of chambers (16) separated by radial walls, thethickness of each radial wall being smaller than the diameter of achannel (15) of the rotating platform (4).
 10. Device according to oneof the preceding claims, characterized in that eluate drain lines (18),preferably in the form of flexible hoses, are attached to the chambers(16) of the eluate collecting ring (5), these hoses first runningdownward, then bending up like a wave in the way of a recumbent “S”,finally bending again downward and, where appropriate, terminating ineluate collection vessels (19), where the highest part of the eluatedrain line (18) preferably is above the level of the eluate collectingring (5).
 11. Device according to one of the preceding claims,characterized in that the inner cylinder of the twin cylinder of therotating body (1) at its lower end is solidly connected with a bottomsupport (11) which in turn rests on the rotating platform (4) and isconnected immovably with it by at least one connecting element, forinstance a bolt, a pin, or a screw (12).
 12. Device according to one ofthe preceding claims, characterized in that it has a number of extensiontubes arranged in the head space above the separation medium andprojecting into the annular gap of the hollow cylinder jacket of therotating body (1), these tubes being connected with the inlet ports forthe liquid supply lines (27 a, 27 c) and, where appropriate, able to beshifted in annular fashion along the periphery of the annular gap abovethe separation medium, and to be fixably arranged at desired angularpositions.
 13. Device according to one of the preceding claims,characterized in that it has means for temperature control and/orthermostatic operation, in particular a heating and/or cooling circuitarranged inside and/or outside of the rotating body (1)
 14. Method forthe purification, separation, and/or concentration of substances bymeans of liquid chromatography using a device for continuous annularchromatography (CAC), in particular a device according to one of theclaims 1 to 13, characterized in that a liquid containing the substancesis applied as sample material in uniform distribution over the entireperiphery of a chromatographic separation medium present in a hollowcylinder jacket of a rotating body, with the stipulation that liquidsfor eluting the substances and conditioning the separation medium areapplied to the separation medium at points situated at predeterminedangular positions and that in the region of these positions apenetration of the sample material into the separation medium ishindered or prevented.
 15. Method according to claim 14, characterizedin that the sample material is supplied via a central inlet into a headspace of the CAC device located above the separation medium andpreferably the entire head space is flooded with the sample material.16. Method according to claim 14 or 15, characterized in that theelution and/or conditioning liquids are applied above the separationmedium at angular positions so selected that no sample material canpentrate into the separation medium between them.
 17. Method accordingto one of the claims 14 to 16, characterized in that the sample materialcontains organic substances, particularly natural or recombinantproteins or mixtures of proteins, and that preferably at least oneconditioning liquid is employed for sanitizing the separation medium.18. Method according to one of the claims 14 to 17, characterized inthat the sample material contains recombinant human factor VIII and withthe method a concentration of rFVIII by a multiple of the startingconcentration is accomplished.